Project Summary Pancreatic cancer (PC) is one of the most lethal malignancies in the United States with a rising incidence and mortality. Gemcitabine (GEM) has been the cornerstone for PC treatment but GEM resistance develops within weeks of chemotherapy initiation, resulting in only a modest impact on survival. Combining GEM with other chemotherapeutic drugs often leads to severe toxicity, without much improvement in survival. It is becoming clear that PC patients exhibit metabolic impairments that can impact their prognosis and survival. In fact, reprogrammed metabolism is a feature of cancer cells and there is an escalated interest in targeting metabolic pathways as a way to improve therapy response. To this end, glutamine metabolism has been widely studied to target cancer cell proliferation. However, the role of other amino acids in modulating PC pathogenesis is still unclear. Our preliminary data provide compelling evidence that serum His level is lower and tissue histidine ammonia lyase (HAL) expression is higher in mice exhibiting PC and human PC patients, compared to controls. Our studies in PC cell-lines showed that His induces cytotoxicity, with a concomitant increase in ammonia production. Moreover, His in combination with GEM exerted a greater cytotoxicity accompanied by a greater reduction in IGF- 1R/mTOR/S6K signaling compared to individual treatments. Therefore, we hypothesize that His and HAL are novel regulators of PC pathogenesis and that targeting His metabolism is a promising approach to enhance the anti-cancer effects of GEM. We will employ nutritional, pharmacological, and metabolic approaches to determine the role and mechanisms by which altered His metabolism regulates the response of PC to GEM therapy. We propose two specific aims: In Specific Aim 1, we will determine the mechanisms by which histidine in combination with gemcitabine exerts enhanced cytotoxic effects against PC cell-lines and PC organoids in vitro. In Specific Aim 2, we will evaluate the therapeutic efficacy of His in combination with GEM in orthotopic and genetic mouse models of PC. Altogether, the studies will uncover the role of His in modulating PC cell metabolism, identify potential mechanisms, and set the stage for future therapeutic discoveries.